During primary infection, herpesviruses establish a latent infection that allows the virus to persist in the host for life. It was recently discovered hat certain herpesviruses integrate their genome into the telomeres of host chromosomes during latency, while most others maintain their genome as an episome in the nucleus. One of these viruses is human herpesvirus 6 (HHV-6), which is associated with a spectrum of diseases including seizures, encephalitis, and graft rejection in transplant patients. However, there are critical gaps in our knowledge regarding the molecular mechanisms of herpesvirus infections, including the mechanisms by which virus gene expression and DNA integration is controlled as well as the events required for virus reactivation. To address these critical gaps in our knowledge, we propose to use our in vitro infection system for HHV-6 integration, latency and reactivation coupled with functional genomics to determine the fate of the virus genome in these processes. We will first determine the viral genomic signatures during the establishment of latency and integration by measuring differential gene expression, chromatin modification and integration events during the transition from lytic to latent infection (Specific Aim 1). Next, we ill then investigate the viral and cellular determinants involved in virus reactivation. Upon treatment of latently infected cells with histone deacetylase inhibitor drugs (HDACi) in vitro, only in a fraction of the cells reactivate. We therefore hypothesize that virus-reactivating cells have a characteristic gene expression response to drug treatment, which will reveal cellular and viral genes that participate in either maintenance of latency or virus reactivation. We will define this drug response by treating cells with HDACi, isolation of reactivating and non-reactivating cells by FACS and performing the assays described in Aim 1 to determine the signature of reactivation (Specific Aim 2). Finally, we will determine the viral genomic signature of cells from iciHHV-6 patients to obtain an insight into the viral regulation in these individuals and to validae our in vitro model (Specific Aim 3). Our proposed experiments will describe, with high-resolution, the connections between transcription, chromatin regulation and viral DNA integration over the course of initial infection, establishment of latency and reactivation. Successful completion of our specific aims will provide knowledge required to understand how transcription from the integrated latent virus genome is controlled, thereby providing possible therapeutic targets to treat viral complications during transplantation.